DNA probes are established tools for sequence detection and quantification. Efficiency and accuracy of sequence detection depend on both hybridization specificity of the probe and its sensitivity. Fluorescence is one the most common ways to detect the hybridization event. Methods that use fluorescent probes for nucleic acid detection are based either on a hybridization-triggered fluorescence of intact probes (e.g., molecular beacons, and linear probes, see U.S. Pat. No. 6,030,787, U.S. Pat. No. 6,214,979, U.S. Pat. No. 5,925,517, U.S. Pat. No. 5,723,591 and U.S. application Ser. No. 10/165,410) or on a quenched-fluorescence release of a probe digested by DNA Polymerase (e.g., methods using TAQMAN®, MGB TAQMAN®; MGB is a trademark of Epoch Biosciences). For those methods utilizing TAQMAN® and MGB TAQMAN® the 5′-exonuclease activity of the approaching DNA Polymerase cleaves a probe between fluorophore and quencher thus releasing fluorescence. These types of probes are suitable for real-time DNA amplification detection. The first type of probes, however, can also be applied for post-amplification sequence detection (scatter plot analysis or thermal melt) or in a solid phase format. Solid phase immobilized quenched fluorescent probes have been disclosed in, for example, U.S. Pat. Nos. 5,876,930 and 6,596,490, and have been used to detect nucleic acid targets. The disclosed hybridization probes have either low signal to background ratios or slow hybridization rates (Xiaohong Fang et al. J. Am. Chem. Soc. 1999, 121: 2921-2922) and unstable, temperature dependent background fluorescence (fluorescence in unhybridized state), which makes them unsuitable for real-time amplification applications.
What is needed in the art are new oligonucleotide probes with high sequence specificity, improved sensitivity (signal to background ratio), stable (temperature independent) background signal and fast hybridization rates. It is also needed that the probes can be readily prepared with a variety of natural and unnatural nucleotides, fluorescent labels and in various lengths. The present invention provides such probes, as well as methods for their preparation and use.